Relative perfusion, pO2, and bioreduction was measured in vivo in tissues in mice by following changes in the intensity and shape of the EPR signals from nitroxides injected directly into the tissues, using low frequency (1.1 GHz) localized EPR spectroscopy. The blood levels of the nitroxides also were followed by placing the tails of the mice in the cavity of a high frequency (9.5 GHz) EPR spectrometer. Using normal and blood flow restricted gastrocnemius muscles it was shown that the decrease of the EPR signals of nitroxides in tissues usually is due mostly to perfusion which redistributed the nitroxides. Bioreduction of the nitroxides was a less important factor. A permanently charged nitroxide was the best indicator for these effects. Changes in pO2 were reflected by changes of the linewidth; only a perdeuterated nitroxide with a narrow line was an adequate indicator for this parameter. We applied this technique to follow both perfusion and oxygen concentration in experimental murine tumors (MTG-B and RIF-1), to determine the feasibility of differentiating both components in these relatively hypoxic model tumor systems, using in vivo EPR spectroscopy. In these systems, increased bioreduction also occurred. By use of different nitroxides and different conditions, the relative effects of all three processes couldbe followed. We also found that the paramagnetic properties of the nitroxides could be used to follow their changes in concentration by affecting (enhancing) T1-weighted MR images, by this means heterogeneity in perfusion in individual tumors was demonstrated. We conclude that the use of magnetic resonance studies of locally injected nitroxides can provide data on relative changes in all three parameters (perfusion, pO2, and bioreduction) and, under appropriate circumstances, potentially also can provide quantitative data.